This invention relates to materials and methods for detecting nucleic acids in samples and, more particularly, to solid phase assays wherein transponders are associated with the beads constituting the solid phase, nucleic acid probes are bound to the surface of the particles and data concerning the assay is encoded on the transponders.
Solid phase assays have been used to determine the presence of nucleic acids, including deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and their modified forms. Solid-phase assays can be applied to nucleic acids either in simple buffers, or in biological fluids, such as blood, serum, plasma, saliva, urine, tissue homogenates, and many others.
In solid phase assays, small beads, or microparticles, are used as the solid phase to capture the analyte. Solid-phase microparticles can be made of different materials, such as glass, plastic, latex, depending on the particular application. Some beads are made of ferromagnetic materials to facilitate their separation from complex suspensions or mixtures.
In conventional solid-phase assays, the solid phase mainly aids in separating molecules that bind to the solid phase from molecules that do not bind to the solid phase. Separation can be facilitated by gravity, centrifugation, filtration, magnetism, immobilization of molecules onto the surface of the vessel, etc. The separation may be performed either in a single step in the assay or, more often, in multiple steps.
Often, there is a need to perform two or more different assays on the same sample, most of the time in a single vessel and at about the same time. Such assays are known in the art as multiplex assays. Multiplex assays are performed to determine simultaneously the presence or concentration of more than one molecule in the sample being analyzed, or alternatively, several characteristics of a single molecule, such as, the presence of several epitopes on a single protein molecule.
One problem with conventional multiplex assays is that they typically cannot detect more than about five analytes simultaneously, because of difficulties with simultaneous detection and differentiation of more than about five analytes. In other words, the number of different analytes that may be assayed in a single solid phase assay is limited by the solid phase.
The present invention overcomes many of these problems by employing transponders associated with the solid phase beads to index the particles constituting the solid phase. Thus, each individual solid phase particle can be assigned a unique index number electronically encoded inside the particle, that can be retrieved at any time, e.g., at one time during the assay, at multiple times during the assay, or continuously during the assay. The index number may define the nucleotide sequence of the oligonucleotide deposited on the surface of the particle, the catalog number of a DNA fragment deposited on the particle, index numbers of chemical steps which were involved in the chemical synthesis of an oligonucleotide bound to the particle, or some other relevant characteristics of the deposited molecules.
In an electronically-indexed multiplex assay of this invention, two or more classes of transponders, each encoded with a different index number and constructed to bind a different nucleic acid sequence, are incubated with the sample in a single vessel. After necessary washes, incubations and additions are performed, the solid phase is analyzed to detect a label indicative of binding of nucleic acid in the sample to the oligonucleotide on the transponder, such as fluorescence, color, radioactivity or the like. Solid phase analysis is either preceded or followed by decoding of the index numbers programmed on the transponders.
Determination of the label and decoding of the memory of the transponder can be done manually on two different instruments, such as a fluorometer and a dedicated scanner, although a single automated instrument that would perform both functions may be used. Such an instrument can be a modified fluorometer in which the scanner is mounted in the proximity of the fluorometer readout window, and reading the sample fluorescence and decoding the transponder are coordinated by a central computer. In addition, such an instrument can be equipped with an automated transport system for transponders.
In one aspect, the present invention provides an electronically-indexed solid phase particle for use in solid phase assays for nucleic acids, comprising a transponder and a nucleic acid sequence attached to the transponder.
In another aspect, the present invention provides a method for detecting nucleic acids in a sample, using solid phase particles having transponders.
In another aspect, the present invention provides kits for detecting nucleic acids in samples, comprising assay vessels, at least one transponder having a nucleic acid probe bound to the transponder, and a labelled reagent to detect binding of sample nucleic acids to the probe.